Monoclonal antibodies to human influx peptide transporter

ABSTRACT

Monoclonal antibodies which react with the human influx peptide transporter are provided. The human influx peptide transporter-reactive antibodies are useful in the purification of influx peptide transporter and in immunoassays to identify agents taken up into the cell by the human influx peptide transporter mechanism. The monoclonal antibodies are also useful for the detection and treatment of carcinomas derived from the gastrointestinal tract and pancreatic duct.

BACKGROUND OF THE INVENTION

Peptides are transported in and out of human cells by several differenttransport mechanisms. One influx peptide transporter has been located inthe brush border of the epithelial cells of the intestinal mucosa.Properties of this intestinal transporter (hereinafter "influx peptidetransporter") have been studied in situ in intestinal mucosapreparations and in vitro with brush border membrane vesicles, isolatedenterocytes, and cell culture.

Many different solutes, including small peptides, antibiotics, oralangiotension converting enzyme (ACE) inhibitors, and oral renininhibitors are transported into the cytoplasm of the enterocyte by theinflux peptide transporter. (e.g., Ganapathy and Leibach, 1991, Curr.Biol. 3:695-701; Okano et al., 1986, J. Biol. Chem. 261:14130-14134;Nakashima et al., 1984, Biochem. Pharm. 33:3345-3352). The influxpeptide transporter plays a pivotal role in the absorption of certainoral drugs, including β-lactams and ACE inhibitors. Out of 27 β-lactamantibiotics examined, the influx peptide transporter was able todistinguish between those that are orally absorbed in humans and thosethat are not (Tabas et al., 1991, 31st Interscience Conference onAntimicrobial Agents and Chemotherapy Abstract No. 164). Moreover, theinflux peptide transporter has been demonstrated to transport a numberof oral β-lactam antibiotics but not parenteral β-lactam antibiotics instudies using human intestinal Caco-2 cells and rabbit intestinalbrush-border membranes (Dantzig et al., 1992, Biochim, Biophys. Acta1112:167-173; Dantzig et al., 1992, 32nd Interscience Conference onAntimicrobial Agents and Chemotherapy, Anaheim, Calif., Abstract No.1460; Snyder et al., 1992, 32nd Interscience Conference on AntimicrobialAgents and Chemotherapy Abstract No. 1461; Okano et al., 1986, J. Biol.Chem. 261:14130-14134.) Similar studies have been conducted to examinethe ability of the influx peptide transporter to predict which ACEinhibitors are orally absorbed (Friedman and Amidon, 1989, Pharm. Res.6:1043-1047).

Influx peptide transporter activity has been identified as a 127,000dalton membrane protein from rabbit intestinal mucosa by photoaffinitylabeling methods employing radiolabeled penicillin or a radiolabeledcephalexin analog (Kramer, 1987, Biochim. Biophys. Acta 905:65-74;Kramer et al., 1988, Biochem. Pharmacol. 37:247-2435). A purified127,000 dalton protein from rabbit intestinal mucosa preparationsreconstituted into liposomes resulted in binding and transportactivities, and polyclonal antibodies to this putative transporter wereprepared (Kramer et al., 1990, Biochim, Biophys. Acta 1030: 50-59).Monoclonal antibodies (MAbs) reactive with human influx peptidetransporter have not been previously described.

Proteins that are expressed in discrete locations in the body can beused as markers to determine the origin of cells or tissues. Forexample, carcinoembryonic antigen is a protein situated only in thecolon and is used as a marker for colon tumor cells (Shrively, 1985,Crit. Rev. Oncol. Heamatol. 2:355-399). MAbs raised to unique proteins,such as the human influx peptide transporter, can be used to identifytumors and metastases that originate from a tissue that expresses thatantigen. This may be done as a diagnostic in the laboratory or may beused in vivo with an imaging agent.

SUMMARY OF THE INVENTION

The present invention provides monoclonal antibodies (MAbs),immunoreactive fragments or recombinants thereof, reactive with humaninflux peptide transporter, a protein of approximately 120,000 daltonmolecular weight.

The present invention also provides a method for preparing a MAb that isreactive with human influx peptide transporter comprising the steps of:

(a) immunizing an immunocompetent mammal with a source of the humaninflux peptide transporter;

(b) fusing lymphocytes of the immunized immunocompetent mammal withmyeloma cells to form hybridoma cells;

(c) screening the hybridoma cells of step (b) for influx peptidetransporter reactivity;

(d) cloning human influx peptide transporter-reactive hybridomas of step(c);

(e) culturing an influx peptide transporter-reactive hybridoma in amedium to secrete said MAb; and

(f) recovering the MAb from the culture supernatant.

The present invention further provides a process for preparing ahybridoma that produces a MAb reactive with human influx peptidetransporter comprising the steps of:

(a) immunizing an immunocompetent mammal with the human influx peptidetransporter;

(b) obtaining lymphocytes from the immunized mammal;

(c) fusing the lymphocytes with myeloma cells to produce hybridomacells; and

(d) cloning a hybridoma cell that produces human influx peptidetransporter reactive MAbs.

In a further embodiment, the invention provides a method of determininguptake of an agent into a cell by a protein reactive with a human influxpeptide transporter-reactive MAb, immunoreactive fragments orrecombinants thereof, comprising:

(a) contacting a human influx peptide transporter-reactive MAb,immunoreactive fragments or recombinants thereof, with a cell that hasinflux peptide transporter activity, in an aqueous solution underconditions that allow the MAb to bind to the cell;

(b) adding the agent to be tested to said solution; and

(c) determining whether transport of the agent into the cell isdecreased by the presence of the MAb.

In still another embodiment, the invention provides a method ofisolating human influx peptide transporter comprising:

(a) immobilizing a MAb, immunoreactive fragments or recombinantsthereof, reactive with human influx peptide transporter onto a surface;

(b) contacting said immobilized MAb with a mixture containing humaninflux peptide transporter under conditions that allow the human influxpeptide transporter to bind to the immobilized MAb;

(c) separating immobilized MAbs that are bound to human influx peptidetransporter from said mixture; and

(d) recovering the human influx peptide transporter by removing thehuman influx peptide transporter from the MAb.

The invention also provides a method of identifying human influx peptidetransporter in a biological sample comprising:

(a) contacting the sample with a MAb, immunoreactive fragments orrecombinants thereof, reactive with the human influx peptidetransporter;

(b) determining the level of binding of said MAb, immunoreactivefragments or recombinants thereof to the sample; and

(c) comparing the amount of the MAb, immunoreactive fragments orrecombinants thereof, bound to substances present in the sample to acontrol sample or to a predetermined base level, so that a bindinggreater than the control level is indicative of the presence of thehuman influx peptide transporter in a biological sample.

The invention further provides a method of diagnosing a humangastrointestinal or pancreatic duct carcinoma or metastases therefromcomprising:

(a) obtaining a body sample from a patient;

(b) contacting the body sample material with a MAb reactive with thehuman influx peptide transporter, immunoreactive fragments orrecombinants thereof;

(c) determining the level of binding of said MAb, immunoreactivefragments or recombinants thereof to the body sample material; and

(d) determining the level of binding of a MAb reactive with human influxpeptide transporter to a body sample known to be free of humangastrointestinal or pancreatic duct carcinoma or metastases therefrom toestablish a control;

(e) comparing the amount of the MAb, immunoreactive fragments orrecombinants thereof bound to substances present in the body sample instep (c) with the amount of the MAb, immunoreactive fragments orrecombinants thereof bound to substances present in the control bodysample of step (d), a binding level greater than the control level beingindicative of the presence of human gastrointestinal carcinoma orpancreatic duct carcinoma or metastases therefrom.

In another embodiment the invention provides a method for diagnosing thepresence of a human gastrointestinal or pancreatic duct carcinoma ormetastases therefrom comprising:

(a) administering to a patient MAb reactive with the human influxpeptide transporter, immunoreactive fragments or recombinants thereof,conjugated to an imaging marker; and

(b) exposing the patient to a means for detecting said imaging marker toidentify the presence of the imaging marker in order to detect primaryhuman gastrointestinal or pancreatic duct carcinoma or metastatic sitestherefrom in a patient.

In another embodiment, the invention provides a method of treating apatient afflicted with human gastrointestinal carcinoma or metastasestherefrom or pancreatic duct carcinoma or metastases therefrom,comprising administering to said patient, a therapeutically effectiveamount of a MAb, immunoreactive fragments thereof or recombinantsthereof reactive with human influx peptide transporter, conjugated to atherapeutic agent.

In another embodiment, the invention provides pharmaceuticalcompositions comprising MAbs, immunoreactive fragments or recombinantsthereof, reactive with human influx peptide transporter.

DESCRIPTION OF THE FIGURES

FIG. 1 is an immunoblot analysis of membranes from human tumor celllines with MAb PTC13G6 performed as described in Example 6. Lane 1 isCaco-2 cells (human colon cells); lane 2 is HT-29 cells (human coloncells); lane 3 is COLO 320 cells (human colon cells); lane 4 CCRF-CEMcells (human lymphoblast cells); lane 5 is IM-9 cells (human lymphoblastcells); lane 6 is SK-N-MC (human neuroblastoma cells); lane 7 is U-373cells (human glioblastoma cells). The arrow in FIG. 1 indicates the bandfor the human influx peptide transporter, an approximately 120,000dalton protein.

FIG. 2 shows the effect of cephalexin uptake by Caco-2 cells in thepresence or absence of MAb PTC13G6.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, inter alia, MAbs which react with thehuman influx peptide transporter, a protein implicated in the transportof various substrates into the cell. The MAbs of this invention areunique in that they inhibit the uptake of substrates by the human influxpeptide transporter. MAbs PTC12D8, PTC13G6, and PTC15F11, are reactivewith human influx peptide transporter and are illustrative of the MAbsof this invention. The hybridomas that produce MAbs PTC12D8, PTC13G6,and PTC15F11, are designated hybridoma PTC12D8, hybridoma PTC13G6, andhybridoma PTC15F11, respectively. Hybridomas MAbs PTC12D8, PTC13G6, andPTC15F11, were deposited with the American Type Culture Collection(ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, on Feb. 17, 1993,and are available under accession numbers HB 11271, HB 11272, and HB11273, respectively.

Human influx peptide transporter-reactive MAbs can be prepared by:immunizing an immunocompetent mammal with a protein source containingthe human influx peptide transporter; fusing a lymphocyte obtained fromthe immunized immunocompetent mammal with an appropriate myeloma cellfusion partner to form a hybridoma; screening the resultant hybridomasfor influx peptide transporter reactivity; and cloning the influxpeptide transporter specific hybridomas by limiting dilution or similartechniques. These and other techniques for the preparation of MAbs areknown in the art (see Rice, et al., Proc. Natl. Acad. Sci. USA,79:7862-7865 (1982); Kurokawa, et al., Nucleic Acids Res., 11:3077-3085(1983); Oi, et al., Proc. Natl. Acad. Sci. USA, 80:825-829 (1983); Boss,et al., Nucleic Acids Res. 12:3791-3806 (1984); Boulianne, et al.,Nature 312:643-646 (1984); Cabily, et al., Proc. Natl. Acad. Sci. USA,81:3273-3277 (1984); Kenten, et al., Proc. Natl. Acad. Sci. USA,81:2955-2959 (1984); Liu, et al., Proc. Natl. Acad. Sci. USA,81:5369-5373 (1984); Morrison, et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984); Neuberger, et al., Nature, 312:604-608 (1984);Potter, et al., Proc. Natl. Acad. Sci. USA, 81:7161-7165 (1984);Neuberger, et al., Nature, 314:268-270 (1985); Jones, et al., Nature(London), 321:522-525 (1986); Oi, et al., BioTechniques, 4:214-221(1986); Sahagan, et al., J. Immunol., 137:1066-1074 (1986); Sun, et al.,Hybridoma 5 (Suppl. I):S17-S20 (1986) Sun, et al., Proc. Natl. Acad.Sci. USA, 84:214-218 (1987); Selected Methods In Cellular Immunology,Mishell and Shiigi, Eds. (W. H. Freeman and Company, New York, N.Y.,1980); and Methods In Enzymology, Volume 73 Part B, Langone and Vunakis,Eds., (Academic Press, Inc., New York, N.Y., 1981), all of which arespecifically incorporated herein by reference.

Numerous species, including rats, mice, rabbits, and goats, are usefulas a source of immunized lymphocytes for the fusion protocols utilizedto generate hybridomas. A/J mice are especially preferred as the sourceof the immune cells for production of human influx peptide transporterspecific antibodies.

The MAbs of the present invention can be altered to a chimeric form bysubstitution, e.g., human constant regions (F_(c) domains) for mouseconstant regions by recombinant DNA techniques known in the art asdescribed in the above cited references. These F_(c) domains can be ofvarious human isotypes, i.e., IgG₁, IgG₂, IgG₄ or IgM.

In addition, the MAbs of the present invention can be altered toaffinity modified forms, avidity modified forms, or both, by alteringbinding sites or altering the hinge region using recombinant DNAtechniques known in the art as exemplified in the above citedreferences.

The recombinant antibody forms can also be fragmented to produceimmunoreactive fragments F(ab')₂, F(ab') or F(ab) in the same manner asdescribed above in which the monoclonal antibody of the presentinvention is fragmented.

Accordingly, as used herein, the expression "recombinant antibodies"collectively includes chimeric/recombinant forms of the monoclonalantibodies of the present invention wherein the Fc domain is substitutedfor an Fc domain of another species or isotype; affinity modified formsof the monoclonal antibodies of the present invention wherein thebinding sites are altered; avidity modified forms of the monoclonalantibodies of the present invention wherein the hinge regions arealtered; immunoreactive fragments of the foregoing and combinationsthereof.

The MAbs of the present invention can be produced in large quantities byinjecting a hybridoma producing monoclonal antibodies of the presentinvention into the peritoneal cavity of pristane-primed mice, and afteran appropriate time (about 1-2 weeks), harvesting ascites fluid from themice. This method yields a very high titer of homogeneous monoclonalantibody. MAbs are isolated therefrom by methods well known in the art(see Stramignoni, et al., Intl. J. Cancer 31:543-552 (1983)).Alternatively, MAbs can be produced by culturing a hybridoma capable ofproducing MAbs of the present invention in vitro and isolating secretedMAbs from the cell culture medium by methods known in the art (seeColcher, et al., Proc. Natl. Acad. Sci. USA 78:3199-3203 (1981)).

To construct hybridomas secreting human influx peptide transporterspecific antibodies, a mouse is immunized with a cell or cellpreparation which possesses human influx peptide transporter activity.For example, a membrane sample from the Caco-2 cell line, prepared asdescribed in Example 1, can be used as the immunogen to produce theMAbs. The Caco-2 cell line is a human colon adenocarinoma cell line thathas been shown to take up antibiotics by an influx peptide transporter(Dantzig and Bergin, 1990, Biochim. Biophys. Acta 1027:211-217; Dantziget al., 1992, Biochim. Biophys. Acta 1112:167-173). Caco-2 cells areavailable from the American Type Culture Collection (ATCC), 12301Parklawn Drive, Rockville, Md. 20852-1776, under accession number ATCCHTB37.

Preferably, a final immunization is carried out by administering aCaco-2 cell membrane preparation directly into the peritoneal cavity ofthe mouse being immunized to enhance the immune response (J. Immunol.Methods 70:39-43, 1984), and obtaining splenocytes from the spleen forfusion with myeloma cells. The hybridomas thus obtained are screened bymeasuring antibody titer in a culture supernatant of the hybridoma.Example 3 of the present disclosure teaches a method for determiningwhether a hybridoma supernatant reacts with human influx peptidetransporter. Antibody-producing hybridomas are then cloned to establishhybridoma cells lines that secrete a MAb specific to the human influxpeptide transporter.

MAbs to the human influx peptide transporter are useful in a method forrapidly identifying agents that are transported into humangastrointestinal cells by this mechanism. Transport of an agent by thismechanism is believed to be a predictor of oral bioavailability. Oralbioavailability is a highly desired property of many medications.Determination of the oral bioavailability of a drug at an early stage ofdevelopment would be particularly advantageous. Presently, drugs areinitially evaluated for oral bioavailability in animal models. Thisprocess requires selection of only a few compounds whose synthesis mustbe scaled up to be evaluated in these models. This process is timeconsuming, laborious, and expensive. Further, there are many examples ofchemical agents that are well absorbed in animal models but not absorbedby humans.

The availability of a family of human influx peptide transporterspecific antibodies enables the development of numerous assay systemsfor characterizing therapeutic agents, such as small, hydrophiliccompounds, that can be transported into human gastrointestinal orpancreatic duct cells by the human influx peptide transporter. The MAbsof this invention are useful in that they bind the human influx peptidetransporter antigen and inhibit human influx peptide transporterfunction. This feature makes the MAbs of this invention especiallyuseful in assays for determining whether an agent is taken up into thecell by the human influx peptide transporter. As indicated above, uptakeof a test agent by this mechanism indicates potential for oralabsorption in vivo. Such an assay measures whether uptake into the cellof a particular compound can be decreased or blocked by a MAb of thisinvention. Decreased or blocked uptake indicates that the test compoundis taken up into the cell by the influx peptide transporter.

As described above, the availability of the MAbs of this invention allowthe facile preparation the human influx peptide transporter insubstantially pure form. After the human influx peptidetransporter-reactive MAb has been prepared and purified, the MAb can beimmobilized on a surface comprising an insoluble matrix, for examplepolysaccharides, synthetic polymers, or inorganic materials, to preparean affinity absorbent. The affinity absorbent can then placed in achromatography column to prepare an affinity column. As an activatedcarrier material cyanogen bromide activated Sepharose 4B (Pharmacia),Affigel 10 (Bio-Rad), Affiprep 10 (Bio-Rad), or the like may be used.The affinity absorbent can then be brought into contact with a humaninflux peptide transporter-containing material, such as a cell membranepreparation of Caco-2 cells. The absorbed human influx peptidetransporter is than eluted by an appropriate elution means such as achange in pH, a change in polarity, protein denaturing agent, chaotropicion or the like. Where the human influx peptide transporter may beinactivated by an eluate, the eluate should be immediately neutralizedor dialyzed.

The human influx peptide transporter is located in the brush border ofthe intestinal mucosa. MAbs to the human influx peptide transporter arealso reactive with tissue from the pancreatic duct. The unique locationof the human influx peptide transporter provides additional utilitiesfor the MAbs of the present invention. For instance, MAbs,immunoreactive fragments or recombinants thereof, reactive with humaninflux peptide transporter can be used either alone, or in combinationwith other antibodies, in in vitro diagnostic assays. For example, bodysamples of patients can be analyzed using labeled MAbs of this inventionfor the detection of the antigen with which the MAb react. In vitrodiagnostic assays for detection of primary human carcinomas ormetastases therefrom by detecting the reactive antigen in body fluids ofpatients using the monoclonal antibodies of the present invention,immunoreactive fragments or recombinants thereof are described ingreater detail below.

The body fluid obtained from a patient is contacted with MAbs of thepresent invention, immunoreactive fragments or recombinants thereof. Adiagnosis is then made by determining the amount of MAb, immunoreactivefragment or recombinants thereof binding to substances present in thebody fluid and comparing the amount of MAb bound to the body fluidsubstances to a predetermined base level. This base level may bedetermined by the level of binding of a MAb reactive with human influxpeptide transporter to a body sample known to be free of humangastrointestinal or pancreatic duct carcinoma or metastases therefrom.The detection of bound MAbs exceeding the base level is indicative ofthe presence of a human gastrointestinal or pancreatic duct carcinoma ormetastases therefrom.

Any body sample suspected of containing the reactive antigen such asblood (serum or plasma), sputum, ascites fluids, pleural effusions,urine and/or biopsy specimens can be used for in vitro diagnosticmethods. Serum, plasma, and urine are the more preferred body fluids foruse in the practice of the invention.

The amount of antibody bound to substances in the body fluid can bedetermined by means of immunochemical assays by means such as thosedescribed, or example, in Klug, et al., Cancer Res. 44:1048-1053 (1984);Klug, et al., Int. J. Cancer 38:661-669 (1986); Herlyn, et al., J. Clin.Immunol. 2:135-140 (1982); Metzgar, et al., Proc. Natl. Acad. Sci. USA81:5242-5246 (1984); Papsidero, et al., Cancer Res. 44:4653-4657 (1984);Hayes, et al., J. Clin. Invest. 75:1671-1678 (1985); Killian, et al.,Cancer Res. 45:886-891 (1985); Hedin, et al., Proc. Natl. Acad. Sci. USA80:3470-3474 (1983); Pekary, et al., Clin. Chem. 30:1213-1215 (1984);Bast, et al., New England J. Med. 309:883-887 (1983); and Bellet, etal., Proc. Natl. Acad. Sci. USA 81:3869-3873 (1984), the disclosures ofall of which are specifically incorporated herein by reference.

The MAbs of the present invention, immunoreactive fragments orrecombinants thereof, can also be used either alone, or in combinationwith other antibodies in in vivo diagnostic assays such as diagnosticimaging or use with an intraoperative hand-held gamma detecting probe.For example, the MAbs, immunoreactive fragments or recombinants thereof,conjugated to an imaging marker, may be used for the in situ detectionof gastrointestinal or pancreatic duct carcinoma lesions. In vivodiagnostic assays for detection of human gastrointestinal or pancreaticduct carcinomas or metastases therefrom using the MAbs of the presentinvention, are described in more detail below.

In such diagnostic assays, MAbs of the present invention, includingimmunoreactive fragments or recombinants thereof and an imaging markerare administered to a patient. The marker may be conjugated to theantibody before administration. Alternatively, subsequent administrationof the marker or linker conjugated marker after administration of themonoclonal antibody is acceptable. The presence of the imaging marker inthe patient is detected by exposing the patient to an appropriate meansfor detecting the particular marker.

Administration and detection of the antibody-imaging marker conjugate aswell as methods of conjugation of the antibody to the imaging marker areaccomplished by methods known to those skilled in the art, as described,for example, in Goldenberg, et al., New England J. Med. 298:1384-1388(1978); Goldenberg, et al., J.A.M.A. 250:630-635 (1983); Goldenberg, etal., Gastroenterol. 84:524-532 (1983); Siccardi, et al., Cancer Res.46:4817-4822 (1986); Epenetos, et al., Cancer 55:984-987 (1985);Philben, et al., Cancer 57:571-576 (1986); Chiou, et al., Cancer Res.45:6140-6146 (1985); Hwang, et al., J. Natl. Cancer Inst. 76:849-855(1986); Colcher, et al., Cancer Res. 43:736-742 (1983); Colcher, et al.,Laboratory Research Methods in Biology and Medicine Immunodiagnostics,New York, Alan R. Liss, pp. 215-258 (1983); Keenan, et al., J. Nucl.Med. 25:1197-1203 (1984); Colcher, et al., Cancer Res. 47:1185-1189(1987); Esteban, et al., Intl. J. Cancer 39:50-59 (1987); Martin, etal., Curr. Surg. 41:193-194 (1984); Martin, et al., Hybridoma 5:S97-S108(1986); and Martin, et al., Am. J. Surg. 150:672-675 (1985); thedisclosures of all of which are specifically incorporated herein byreference.

The dosage of the MAb imaging agent is a pharmaceutically acceptableamount which will vary depending upon the age and weight of the patient,but generally a one time dosage of about 0.1 to 20 milligrams ofantibody-marker conjugate is sufficient. A more preferred dosage isabout 1.0 to 2.0 milligrams of antibody-marker conjugate.

Imaging markers which can be conjugated to the antibody are known tothose skilled in the art and includes substances which can be detectedby a gamma scanner or hand-held gamma probe, Positron EmissionTomography or the like as described in the references cited above, andsubstances which can be detected by nuclear magnetic resonance imagingusing a nuclear magnetic resonance spectrometer (also described in thereferences cited above).

Examples of substances which can be detected using a gamma scanner orthe like include ¹²⁵ I, ¹³¹ I, ¹²³ I, ¹¹¹ In, and ^(99m) Tc. ¹¹¹ In and^(99m) Tc are preferred due to their low energy and suitability for longrange detection.

An example of a substance which can be detected using a nuclear magneticresonance spectrometer or the like is the nuclear magneticspin-resonance isotope gadolinium (Gd).

MAbs to the human influx peptide transporter can be used in vivo becauseof the unique location of the human influx peptide transporter. Thehuman influx peptide transporter is located in the brush border of theintestinal mucosa and not on the basolateral side of the enterocyte.Because the intestinal mucosa is a tight epithelium which does notpermit solutes to readily pass paracellularly (i.e. between cells), andthe influx peptide transporter faces the lumen of the intestine, MAbsadministered intravenously or intraperitoneally would not be expected tohave ready access to the antigen located on the brush border. Becausetumor cells do not form a tight epithelium, it is likely that theantigen would be randomly distributed along the cell surface.Consequently, the MAb would localize predominately to tumors expressingthe influx peptide transporter.

The MAbs of the present invention, immunoreactive fragments orrecombinants thereof, can also be used either alone, or in combinationwith other antibodies, in immunohistopathology or immunocytochemistryassays for the diagnosis of human carcinomas or metastases therefrom.For example, the MAbs of the present invention, are added to a slidecontaining a 5 micron section of a biopsy specimen (forimmunohistochemistry) or cells (for immunocytochemistry) from body fluid(such as a pleural effusion, ascites sputum, or vaginal fluid). A seriesof linkers (e.g., biotinylated horse anti-mouse IgG followed by avidinDH:biotinylated horseradish peroxidase complex) and dyes (e.g.diaminobenzidine) are then added to the slides to detect binding of themonoclonal antibody, immunoreactive fragment or recombinant thereof tocarcinoma cells in the biopsy or body fluid by a color reaction, i.e.,carcinoma cells will look reddish-brown, while normal and benign cellswill look blue (the background stain). Other detection methods may beused with alternate linkers, dyes, and subsequent color reactions (see,for example, Sternberger, L.A., Immunocytochemistry, New York, JohnWiley & Sons, Second Edition, pp. 82-169 (1979)). By this method: (a)carcinoma cells can be detected in biopsy specimens and body fluids asan adjunct to making a diagnosis of cancer, and (b) a differentialdiagnosis can be made. For example, MAb PTC13G6 has been shown to bindhuman tumor cell lines derived from the colon but not to human tumorcell lines derived from the human blood and brain (see FIG. 1). Thus,detection of binding of MAbs of the present invention (includingimmunoreactive fragments or recombinants thereof) would indicate agastrointestinal origin of the primary carcinoma.

The use of immunohistochemistry and immunocytochemistry assays for thediagnosis of cancer or to make differential diagnoses are accomplishedby methods known in the art, as described, for example, in Nuti, et al.,Intl. J. Cancer 29:539-545 (1982); Stramignoni, et al., Intl. J. Cancer31:543-552 (1983); Szpak, et al., Acta Cytologica 28:356-367 (1984);Johnston, et al., Cancer Res. 45:1894-1900 (1985); Szpak, et al., Am. J.Path. 122:252-260 (1986); Thor, et al., J. Natl. Cancer Inst.76:995-1006 (1986); Martin, et al., Am. J. Clin. Path. 86:10-18 (1986);Nuti, et al., Intl. J. Cancer 37:493-498 (1986); Johnson, et al., CancerRes. 46:850-857 (1986); Thor, et al., Cancer Res. 46:3118-3124 (1986);Obuchi, et al., Intl. J. Cancer 38:643-650 (1986); Johnston, et al.,Cancer Res. 46:6462-6470 (1986); and Thor, et al., Cancer Res.47:505-512 (1987), the disclosures of all of which are specificallyincorporated herein by reference.

Labeling of the MAb for use in such assays is carried out byconventional procedures. For the radioimmunoassay, a radioisotope suchas ¹²⁵ I is used. For the enzyme immunoassay, conventional enzymes suchas peroxidase or β-galactosidase are used. For the fluorescentimmunoassay, conventional chromophores can be used as markers. Theseassay methods, and modifications thereof, are well known in the art.Assay systems are discussed further in Methods in Enzymology, Vol. 73,Part B, supra, the contents of which are herein incorporated byreference. Section II of Methods in Enzymology, Vol. 73, Part B, supra,discusses labeling of antibodies and antigens, while Section IV,discusses immunoassay methods.

MAbs to the human influx peptide transporter are also useful in in vivotreatment of human gastrointestinal carcinomas or human pancreatic ductcarcinomas or metastases therefrom. In vivo treatment of humangastrointestinal carcinomas or metastases therefrom using MAbs of thepresent invention, immunoreactive fragments or recombinants thereof isdescribed in greater detail below.

A pharmaceutically effective amount of MAbs of the present invention,immunoreactive fragments or recombinants thereof unconjugated orconjugated to a therapeutic agent is administered to a patient. Methodsof preparing and administering the monoclonal antibody-therapeutic agentconjugate as well as suitable dosages will depend on the age and weightof the patient and the therapeutic agent employed and are known to orreadily determined by those skilled in the art. Representative protocolsare described in the references cited below.

Examples of the monoclonal antibody-therapeutic agent conjugates whichcan be used in therapy include antibodies coupled to radionuclides, suchas ¹³¹ I, ⁹⁰ Y, ¹⁰⁵ Rh, ⁴⁷ Sc, ⁶⁷ Cu, ²¹² Bi, and ²¹¹ At, as described,for example by Goldenberg, et al., Cancer Res. 41:4354-4360 (1981);Carrasquillo, et al., Cancer Treat. Rep. 68:317-328 (1984); Zalcberg, etal., J. Natl. Cancer. Inst. 72:697-704 (1984); Jones, et al., Int. J.Cancer 35:715-720 (1985); Lange, et al., Surgery 98:143-150 (1985);Kaltovich, et al., J. Nucl. Med. 27:897 (1986); Order, et al., Int. J.Radiother, Oncol. Biol. Phys. 8:259-261 (1982); Courtenay-Luck, et al.,Lancet 1:1441-1443 (1984) and Ettinger, et al., Cancer Treat. Rep.66:289-297 (1982), the disclosure of all of which are specificallyincorporated herein by reference. Antibodies coupled to drugs orbiological response modifiers such as methotrexate, adriamycin, andinterferon as described, for example, in Chabner, et al., CancerPrinciples and Practice of Oncology, Philadelphia, Pa., J. B. LippincottCo. Vol. 1, pp. 290-328 (1985); Oldham, et al., Cancer Principles andPractice of Oncology, Philadelphia, Pa. J. B. Lippincott Co., Vol. 2,pp. 2223-2245 (1985); Deguchi, et al., Cancer Res. 46:3751-3755 (1986);Deguchi, et al., Fed. Proc. 44:1684 (1985); Embleton, et al., Br. J.Cancer 49:559-565 (1984) and Pimm, et al., Cancer Immunol. Immunother.12:125-134 (1982), the disclosure of all of which are specificallyincorporated herein by reference. Antibodies coupled to toxins, asdescribed, for example, in Uhr, et al., Monoclonal Antibodies andCancer, Academic Press, Inc., pp. 85-98 (1983), Vitetta, et al., Science219:664-650 (1983), the disclosure of all of which are specificallyincorporated herein by reference. Heterobifunctional antibodies forexample, antibodies coupled or combined with another antibody so thatthe complex binds both to the carcinoma and effector cells, e.g., killercells such as lymphokine activated killer (hereinafter "LAK") (seeRosenberg, et al., Science 223:1412-1415 (1984) or T cells, asdescribed, for example, in Perez, et al., J. Exper. Med. 163:166-178(1986); and Lau, et al., Proc. Natl. Acad. Sci. USA 82:8648-8652 (1985);the disclosures of both of which are specifically incorporated herein byreference; and native, i.e., non-conjugated or non-complexed, antibody,as described in, for example, in Shiloni, et al., J. Immunol.138:1992-1998 (1987); Eisenthal, et al., Cancer Res. 47:2771-2776(1987); Honsik, C. J. et al., Proc. Natl. Acad. Sci. USA 83:7893-7897;Herlyn, et al., Proc. Natl. Acad. Sci. USA 79:4761- 4765 (1982); Schulz,et al., Proc. Natl. Acad. Sci. USA 80:5407-5411 (1983); Capone, et al.,Proc. Natl. Acad. Sci. USA 80:Z328-7332 (1983); Sears, et al., CancerRes. 45:5910-5913 (1985); Nepom, et al., Proc. Natl. Acad. Sci. USA81:2864-2867 (1984); Koprowski, et al., Proc. Natl. Acad. Sci. USA81:216-219 (1984); and Houghton, et al., Proc. Natl. Acad. Sci. USA82:1242-1246 (1985) all of which are specifically incorporated herein byreference.

The monoclonal antibody-therapeutic agent conjugate is delivered to thegastrointestinal carcinoma site where binding occurs, thereby directlyexposing the carcinoma tissue to the therapeutic agent. Previoustherapeutic studies using high doses of monoclonal antibodies such as17-1A, which is reactive with normal gastrointestinal epithelium, haveresulted in no apparent toxicity (see Sears, et al., J. Biol. Resp. Mod.3:138-150 (1984). Furthermore, even where toxicity results in damage,normal gastrointestinal epithelium has been shown to regenerate aftersuch exposure while carcinoma cells do not regenerate.

A pharmaceutical composition comprising the MAbs, immunoreactivefragments or recombinants thereof, of the present invention in apharmaceutically acceptable, non-toxic, sterile carrier such asphysiological saline, non-toxic buffers, and the like, now also becomespossible. The amount of said MAbs in the pharmaceutical compositionshould be sufficient to achieve effective binding with the antigenagainst which said MAbs have specific affinity or neutralizingreactivity. The pharmaceutical composition may be administered by singleor multiple dosage with other adjuvants or additives, if necessary, inany suitable manner to the host in need of said MAbs.

MAbs to the human influx peptide transporter are also useful as tools inthe cloning of cDNA for the influx peptide transporter and relatedgenes. As described in Example 7, these MAbs can be used to detect theexpression of cDNA encoding the human influx peptide transporter. TheMAbs of this invention may also be used to detect expression of proteinswith the same or closely related antigen as the human influx peptidetransporter. Thus, the MAbs of this invention have an additional utilityin that they are useful in the identification of DNA encoding the humaninflux peptide transporter or related genes. Techniques for DNA cloningand expression thereof are described by Maniatis, et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Press, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1989).

The MAbs of this invention can also be used for detecting proteins whichpossess the same or a closely related antigen to the influx peptidetransporter. Also, as indicated in Example 5, the MAbs of this inventionare useful in providing clinical information regarding the tissuedistribution of the influx peptide transporter.

The Examples which follow are intended to further illustrate the presentinvention and are not to be interpreted as limiting on the scopethereof.

EXAMPLE 1 Preparation of Caco-2 Membranes

Caco-2 cells are available from the American Type Culture Collection(ATCC), 12301 Parklawn Drive, Rockville, Md. 20852-1776, under accessionnumber ATCC HTB37. Caco-2 cells were grown in Dulbecco's Modified Eaglemedium (Gibco, Grand Island, N.Y.) containing 10% fetal calf serum(Hyclone) and 1% Minimal Essential Media non-essential amino acidsolution (Gibco) as previously described by Dantzig et al., 1990,Biochim. Biophys. Acta 1027:211-217, the entire contents of which areincorporated herein by reference.

Partially purified Caco-2 membranes were prepared from a 2 to 3 weekpost-confluent culture of Caco-2 cells as follows. A total ofapproximately 4×10⁸ cells (grown as described above) were disruptedusing a glass Dounce homogenizer. Membranes were collected bydifferential centrifugation (Lever, 1977, J. Biol. Chem. 252:1990-1997).After homogenization in 0.25M sucrose, 0.1M Tris, pH 7.5, 0.2 mM CaCl₂,0.04 mg/ml bacitracin, 2 μg/ml aprotinin and 0.02 mMphenylmethanesulfonylfluoride, the sample was centrifuged at 3,000 x gfor 10 minutes. Supernatants were layered over a 35% sucrose gradientand centrifuged at 16,000 x g for 1 hour. The interface was removed, andcentrifuged at 100,000 x g for 60 minutes. Membranes were enrichedapproximately 6-fold in alkaline phosphatase activity measured with analkaline phosphatase assay kit (Sigma Chemical Co., St. Louis, Mo.,Procedure No. DG 1245).

EXAMPLE 2 Preparation of MAbs Against Human Influx Peptide TransporterA) Immunization

Mice (A/J, Jackson Laboratory, Bar Harbor, Me.) were injectedsubcutaneously three times with a 140-250 μg protein sample of theCaco-2 membranes (prepared as described in Example 1B), over a 6 weekperiod. The mice were boosted with an injection of 180 μg protein threedays prior to sacrifice. On day 94 blood was removed from the mice toyield 400 μl of antiserum.

B) Preparation of Myelomas

On day 94 the mice were sacrificed and their spleens were removed.Activated lymphocytes were isolated and fused with mouse myeloma(X63/Ag8.653, ATCC, Rockville, Md.) according to standard hybridomamethods. See generally, Starling et al., Cancer Immunol. Immunother.28:171 (1989).

C) Growth of the Hybridomas

After the fusion, hybridomas were diluted and grown for 23 days inEagle's medium (Gibco) supplemented withhypoxanthine/aminopterin/thymidine selection medium (Sigma Chemical Co.)and gentamicin (25 μg/ml; Sigma) in 96-well microtiter dishes.

EXAMPLE 3 Hybridoma Screening

After 2-3 weeks, approximately 1200 hybridomas were screened for theability to block the accumulation of 1 mM cephalexin into two to threeweek post-confluent Caco-2 cells in substantial accordance with theassay described by Dantzig et al., 1990, Biochim, Biophys. Acta1027:211-217. Cephalexin is available from Eli Lilly and Company,Indianapolis, Ind. Briefly, supernatants from these hybridomas werepooled by rows and by columns yielding 320 pooled supernatants.Confluent Caco-2 cells were washed with Earle's balanced salt solution(Gibco) containing 25 mM HEPES, pH 7.4 (EBSS) and incubated 45 minutesat 37° C., and then the EBSS was removed by aspiration. 300 μl samplesof each pooled supernatant were added to the cells and incubated at 37°C. for 2 hours. Uptake of 1 mM [¹⁴ C]cephalexin (labeled as described byDantzig et al., Biochem. Biophys. Res. Commun. 155:1082-1087) wasmeasured at 37° C. for 1 hour in sodium-free Earle's balanced saltsolution containing 120 mM choline chloride, 25 mM MES(2-(N-morpholino)-ethanesulfonic acid), pH 6.0 (sodium-free,Trans-EBSS). Subsequently cells were washed three times with ice coldEBSS, pH 7.4. Microtiter dishes were screened for radioactivity using anAMBIS (Radioanalytical Imaging System, Model 100, Ambis, San Diego,Calif.). Using this methodology, 24 pooled supernatants were identifiedas reducing uptake.

Samples of individual supernatants (800 μl) from the rows and columnsthat corresponded to the pooled supernatants that reduced [¹⁴C]cephalexin uptake were subsequently screened for inhibition of [¹⁴C]cephalexin uptake. The supernatants of three hybridomas reduced uptakeusing the same protocol described above for the pooled supernatants. Twoinhibited uptake by 60-90% in the initial screen (Hybridoma PTC12D8 andHybridoma PTC13G6 and a third blocked uptake by 20% (Hybridoma MAbPTC15F11).

These hybridomas were subcloned by limiting cell dilutions followed bythe fluorescence activated cell sorting technique reported by Marder etal., 1990 Cytometry, 11:498-505. The isotypes of the MAbs of thisinvention were determined by a commercially available isotyping kit("Mouse Typer", Bio-Rad Labs, Richmond, Calif. 94804). MAbs PTC12D8 andPTC13G6 were found to be an IgG₁ (Kappa) isotype. MAb PTC15F11 was foundto be an IgM (Kappa) isotype.

EXAMPLE 4 Characterization of the Antigen A. Separation of MembraneProteins and Immunoblot Analysis

Ten to fourteen day post-confluent Caco-2 cells were grown in a T-75flask in Dulbecco's Modified Eagle medium (Gibco) at 37° C. Afteraspirating off the growth media, 5 ml of phosphate-buffered saline, pH7.4 (PBS) were added and cells were scraped and removed. The cells werecollected by centrifugation and then resuspended in 1 ml phosphatebuffered saline (PBS) and placed in 1.5 ml microfuge tube. Cells werecollected in a microfuge. Buffer was removed and cells were resuspendedin 200 μl 0.01M Tris-HCl (pH 7.4) 1% Triton X-100 and placed on ice for20 minutes. Cells were drawn in and out of a syringe three times with a25 gauge needle. The lysate was centrifuged at 12,000 x g for 5 minutes.The supernatant containing crude membranes was removed and the proteincontent was determined. An aliquot of the membrane preparationcontaining 100 μg of protein was mixed 1:4 v/v with a 9.2% sodiumdodecyl sulfate (SDS) and then 100 μl of bromophenol blue solutions wasadded. A sample of these membrane proteins was separated by 8-16%SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with a running bufferof 0.25M Tris, 1.92M glycine, 1% w/v SDS at 45 volts for approximately14 hours. The gel was electroblotted onto nitrocellulose paper in 50 mMphosphate, pH 7.4, at 1.6 amps for 3 hours. The paper (blot) was blockedwith 3% bovine serum albumin and 15 mM NaCl, 1 mM EDTA, and 0.1% TritonX-100 in Tris-HCl, pH 7.5 (rinse buffer) at 25° C. The blot was washedtwice with rinse buffer. The blot was incubated in rinse buffercontaining purified mouse anti-human peptide transporter MAb (MAbsPTC13G6, PTC12D8, or MAb PTC15F11; 20 μg/ml) and incubated for 1 hour at25° C. The blot was subsequently washed three times with rinse buffer,and incubated in rinse buffer containing 50 ng/ml goat [¹²⁵ I] antimouseimmunoglobulin G (0.3 μCi/ml) or 32 ng/ml goat [¹²⁵ I] antimouseimmunoglobulin M for 30 minutes at 25° C. The blot was washed fourtimes, air dried, and exposed to film with two intensifying screens at-70° C. overnight. MAbs PTC13G6 and PTC12D8 detected the presence of asingle ˜120 kilodalton protein. MAb PTC15F11 detected no protein band.

Although MAb PTC15F11 detected no band, the MAb does react with thepeptide transporter. The specificity of MAb PTC15F11 is conformationdependent. Protein conformation is altered by electrophoresis andelectroblotting. MAb PTC15F11's specificity for the influx peptidetransporter was measured as follows. An aliquot of the Caco-2 membranepreparation containing 30 μg of protein was added to a mixturecontaining: 50 μl of agarose conjugated to goat anti-mouse antibody (Fcspecific for IgM, Sigma Chemical, St. Louis, Mo.), 4 μg of PTC15F11, 300μl of PBS (pH 7.4) and 2% bovine serum albumin (BSA). The membraneprotein preparation had been precleared by preincubation with the goatanti-mouse immunoglobulin agarose conjugate and then using thesupernatant. This is done to remove any proteins that may bindnon-specifically to the agarose or goat anti-mouse immunoglobulin.

The immunoprecipitation mixture was rocked gently at 4° C. for 16 hours.The agarose pellet was washed 3 times in PBS by centrifugation. Thepellet was resuspended in sodium dodecyl sulfate (SDS) sample buffer(0.125M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 5% 2-mercaptoethanol)then boiled for 5 minutes. An aliquot of the supernatant was loaded ontoan 8-16% SDS-polyacrylamide gel. The proteins were separated byelectrophoresis with a running buffer of 0.025M Tris, pH 8.3, 0.192glycine, 0.1% SDS at 30 mA for 90 minutes.

The gel was electroblotted onto nitrocellulose paper in 12 mM Tris, 96mM glycine, pH 8.3 with 20% methanol at 30 volts for 90 minutes. Thepaper was blocked in 2% BSA in 0.02M Tris, 0.15M NaCl, pH 7.4(Tris-buffered saline, TBS) for 16 hours at 4° C. The blot was thenincubated in TBS containing 0.2% BSA (incubation buffer) and MAb PTC13G6(20 μg/mL) for 2 hours at 25° C. The blot was washed 3 times with TBScontaining 1 mM EDTA and 0.1% Triton X-100 (rinse buffer). The blot wasincubated in incubation buffer containing goat anti-mouseIgG-horseradish peroxidase (Sigma Chemical) at a 1:1000 dilution for 1hour at 25° C. The blot was washed 3 times with rinse buffer and 1 timewith TBS. Tetramethylbenzidine substrate (TMB; Kirkgaard and Perry Labs,Gaitherburg, Md.) was added to the blot and allowed to react for 15minutes at 25° C. The TMB solution was removed and deionized water wasadded immediately to stop the reaction. MAb PTC13G6 detected thepresence of a 120 kilodalton protein following immunoprecipitation withMAb PTC15F11.

B. Purification of MAbs

MAbs PTC12D8 and PTC13G6 were purified as follows. Crude ascites fluidcontaining either PTC12D8 or PTC13G6 was prepared according to standardmethods (Campbell, 1986, Monoclonal Antibody Technology p. 230). Theascites fluid was diluted 1:1 with glycine-OH buffer (1.45M, pH 8.9) andadded to a Sepharose-Protein A column (5 ml bed volume). The column waswashed with 10 bed volumes of the glycine buffer. The MAb was elutedwith 0.04M sodium citrate, pH 3.2. The eluted fraction was neutralizedby adding 0.5M sodium phosphate buffer, pH 7.7, at a ratio of 1:10. TheMAb prep was dialyzed against phosphate buffered saline (PBS), pH 7.2and read at 280 nm to determine concentration.

MAb PTC15F11 was semipurified by precipitating the MAb from crudeascites with 18% sodium sulfate, and then dialyzed against PBS. Thesemipurified preparation was injected into a Zorbax GF-450 sizeexclusion column (DuPont, Wilmington, Del. 1989). Fractions obtainedfrom the largest isolated peak were pooled, and read at 280 nm todetermine concentration.

EXAMPLE 5 Immunohistochemistry

Frozen tissue specimens of normal human tissue (5μ sections) were driedand fixed with formalin in accordance with the method of Wilson et al.,1990, Cancer Research 50:3124-3130. Specimens were incubated with 5μg/ml of MAb PTC13G6 followed by incubation with a secondary goatanti-mouse IgG antibody conjugated to horseradish peroxidase. Anirrelevant, isotype matched antibody, was used for comparison. Analysisrevealed that MAb PTC13G6 was strongly positive to human tissues alongthe gastrointestinal tract (jejunum, duodenum, ileum, and colon) and tothe ducts of the pancreas, and negative to kidney, lung, liver, brain,adrenal gland, and skin. Furthermore, the antigen was expressed on theintestinal (apical) side of the gastrointestinal epithelium. It isnoteworthy that the antigen was absent in kidney tissues since a peptidetransporter is present there (Miyamoto et al., 1985, Biochem. Biophys.Res. Commun. 132:946-953; Skopicki et al., 1991, Am. J. Physiol.261:F670-F678.

EXAMPLE 6 Immunoblot Analysis

Membrane proteins of a variety of human tumor cell lines were preparedin substantial accordance with the method described in Example 1.Membrane proteins were separated by 8% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The gel waselectroblotted onto nitrocellulose paper, blocked with 3% bovine serumalbumin and 0.1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 0.01M Tris-HCl,pH 7.5, and reacted with 20 μg/ml MAb PTC13G6 followed by incubationwith goat anti-mouse [¹²⁵ I]immunoglobulin G (0.3 μCi/ml). The resultsare shown in FIG. 1. Lane 1 is Caco-2 cells (human colon); lane 2 isHT-29 cells (human colon); lane 3 is COLO 320 cells (human colon); lane4 CCRF-CEM cells (human lymphoblast); lane 5 is IM-9 cells (humanlymphoblast cells); lane 6 is SK-N-MC (human neuroblastoma cells); lane7 is U-373 cells (human glioblastoma cells). The arrow in FIG. 1indicates the band for the human influx peptide transporter, anapproximately 120,000 dalton protein. As indicated by FIG. 1, MAbPTC13G6 detected the presence of the human influx peptide transporter inhuman colon tumor cell lines. MAb PTC13G6 did not detect the presence ofthe human influx peptide transporter in human tumor cell linesoriginating from the blood or the brain.

EXAMPLE 7 Inhibition of Cephalexin Uptake

The effect of preincubation of Caco-2 cells with increasingconcentrations of MAb PTC13G6 on 1 mM [¹⁴ C]cephalexin uptake wasmeasured as follows. Growth medium was removed from 14-day oldpost-confluent Caco-2 cells grown in Costar multiwell dishes (24 wells)and replaced with 1.5 ml of diluted supernatant from hybridoma PTC13G6.Cells were incubated for 2 to 2.5 hours, rinsed once with 1 ml of 37° C.Trans-EBSS, pH 6.0. After aspiration, the uptake rate of 1 mM [¹⁴C]cephalexin in Trans-EBSS, pH 6.0 was measured over a 4 minute timecourse in accordance with the method described by Dantzig, 1990, supra.The results of this assay are shown in FIG. 2. The shaded portion ofFIG. 2 represents uptake due to simple diffusion which was estimated bythe portion that could not be inhibited by the presence of 50 mM of thedipeptide glycyl-L-proline.

EXAMPLE 8 Expression Cloning

Plasmid pPSJ179 is approximately 8500 base pairs in length and containsDNA encoding the influx peptide transporter from Caco-2 cells. PlasmidpPSJ179 was constructed by cloning a 3.4 kilobase pair XbaI-HindIII cDNArestriction enzyme fragment that comprises the influx peptidetransporter-encoding DNA into the commercially available vector pRc/RSV(Invitrogen). The 3.4 kilobase pair XbaI-HindIII cDNA restriction enzymefragment was identified with the MAb PTC13G6 by expression cloningessentially as described by Maniatis et al., 1989. Plasmid pPSJ179contains the ampicillin resistance gene for selection in Escherichiacoli, neomycin resistance gene for selection in eukaryotic cells and theinflux peptide transporter gene positioned for expression from the RousSarcoma virus (RSV) promoter. Plasmid pPSJ179 can be isolated fromEscherichia coli K12 DH5α/pPSJ179 using a standard alkaline-SDSprocedure (Maniatis et al., 1989). Escherichia coli K12 DH5α/pPSJ179 wasdeposited with the Northern Regional Research Laboratories (NRRL) inPeoria, Ill. 61604, on Jan. 21, 1993, and is available under accessionnumber NRRL B-21041.

Chinese hamster ovary cells (CHO-K1, ATCC CCL 61) were transfected withplasmid pPSJ179 using a calcium precipitation protocol described in theStratagene human transfection kit (Stratagene Catalog #200285). Thecalcium precipitation protocol transfection method was carried out asfollows. Subconfluent CHO-K1 cells (100 mm culture dish, 1-day postplating) were incubated for 24 hours in a 3% CO₂ incubator at 37° C.with 20 μg of a calcium-precipitated DNA sample. The DNA sample waseither plasmid pPSJ179 or, as control, plasmid pRc/RSV, the vector usedto construct pPSJ179. Subsequently, the cells were grown for 3 days inF12 medium containing 10% fetal bovine serum (Hyclone Laboratories Inc.,Logan, Utah 84321). Afterwards, the medium was replaced with growthmedium containing the selection agent, G-418 sulfate at 300 μg/ml(Gibco, Grand Island, N.Y.) and cells were grown in a 5% CO₂ incubatorat 37° C. for 13 days. Colonies which were selected for further studywere grown for selected time periods in the selection medium in 24 wellplates at 37° C. in a 5% CO₂ incubator. Transfectants were evaluated forexpression of the influx peptide transporter using an enzyme linkedimmunosorbent assay (ELISA) and MAb PTC13G6. The ELISA was performed asfollows. The medium was removed from the 24 well plates that containedmonolayers of the CHO-K1 cells. Two ml of TBS-BLOTTO (TBS 0.02M Tris,0.14M NaCl, pH 7.4) containing nonfat dried milk (0.5%)) were added toeach well and the plates were incubated at room temperature for 30minutes. Twenty μg/ml of MAb PTC13G6, 15 μg/ml of a control monoclonalantibody, or buffer alone, were added to each well in a volume of 250μl. The plate was incubated at room temperature for 45 minutes.Following incubation, the cells were washed one time with TBS-BLOTTO.Goat anti-mouse IgG-horseradish peroxidase (Jackson ImmunoresearchLaboratories, West Grove, Pa.) diluted 1:2000 was added to each well ina volume of 250 μl. The plates were incubated at room temperature for 45minutes. Following incubation, the samples were washed three times withTBS-BLOTTO and then washed two times with TBS. Tetramethyl-benzidinesubstrate (TMB) (Kirkegaard and Perry Labs, Gaithersburg, Md.) was addedto each well in a volume of 250 μl. The samples were incubated at roomtemperature for 20 minutes. One molar H₃ PO₄ was then added to each wellin a volume of 250 μl to stop the reaction. A 100 ml aliquot wastransferred from each well of the 24 well plate to a single well on a 96well plate. Absorbance readings were read at 450 nm on aspectrophotometer. Higher absorbance readings indicate binding of theMAb, and therefore, presence of the influx peptide transporter antigen.Clones that expressed higher levels of the influx peptide transporterantigen than the control were selected for transport studies.

EXAMPLE 9 [¹⁴ C]cephalexin Uptake

Clones selected by ELISA in Example 8 were evaluated for the uptake of 1mM [¹⁴ C]cephalexin. The transfected CHO-K1 cells (˜2.4×10⁴ cells perwell) were grown for 13-17 days in a Costar 24-well plate as describedabove. Confluent cells were washed with Earle's balanced salt solution(Gibco) containing 25 mM HEPES, pH 7.4 (Trans-EBSS) and incubated 45minutes at 37° C., and then the Trans-EBSS was removed by aspiration.The cells were incubated in the presence of 1 mM [¹⁴ C]cephalexin insodium-free Earle's balanced salt solution containing 120 mM cholinechloride, 25 mM MES, pH 6.0 (sodium-free, Trans EBSS) at 37° C. for 2hours. Subsequently, cells were washed with ice cold Trans-EBSS, pH 7.4,lysed in 0.2N NaOH, and an aliquot was removed for scintillationcounting.

A representative transfectant (Clone 9) displayed significantly higheruptake of [¹⁴ C]cephalexin than the control. A further study indicatedthat uptake of 1 mM cephalexin by this transfectant was inhibited by thepresence of 15 mM of bestatin, a dipeptide that is known to compete foruptake by the influx peptide transporter (Saito et al., 1993, Biochem.Pharmacol. 45(3):776-779 and Inui et al., 1992, J. Pharmacol. Exp.Therap. 260:482-486), or 25 mM unlabelled cephalexin. Coincubation ofthe cells with 1 mM [¹⁴ C]cephalexin and bestatin or unlabelledcephalexin decreased drug uptake in the representative transfectant(Clone 9) to the level of the control cells. Moreover, transport of 1 mM[¹⁴ C]cephalexin by the control cells was not inhibited significantly bybestatin or by cephalexin. The results of these studies are provided inTable 1.

                  TABLE 1                                                         ______________________________________                                                        [.sup.14 C]-Cephalexin Uptake                                                 (nmol/mg total cell                                           Sample          protein)                                                      ______________________________________                                        Clone 9         1.11 ± 0.08                                                Clone 9 + bestatin                                                                            0.42 ± 0.08                                                Clone 9 + cephalexin                                                                          0.35 ± 0.09                                                Control         0.55 ± 0.09                                                Control + bestatin                                                                            0.38 ± 0.08                                                Control + cephalexin                                                                          0.31 ± 0.06                                                ______________________________________                                    

We claim:
 1. A method of determining whether an agent is transportedinto a cell by a human influx peptide transporter which is specificallybound by monoclonal antibodies PTC13G6, PTC12D8, and PTC15F11, whereinsaid method comprises:(a) contacting a cell which expresses said humaninflux peptide transporter with a monoclonal antibody, an antibodyfragment or a recombinant antibody which specifically binds said humaninflux peptide transporter and which inhibits the function of the humaninflux peptide transporter, in an aqueous solution under conditions thatallow the monoclonal antibody, antibody fragment or recombinant antibodyto bind to the cell; (b) adding the agent to be tested to said solution;and (c) determining whether transport of the agent into the cell isinhibited by the presence of the monoclonal antibody, antibody fragmentor recombinant antibody.
 2. The method of claim 1 wherein the monoclonalantibody is selected from the group consisting of PTC13G6, PTC12D8, andPTC15F11.
 3. The method of claim 2 wherein the monoclonal antibody isPTC13G6.